Aberrant Platelet Aggregation as Initial Presentation of Essential Thrombocythemia: Failure of Entero-Coated Aspirin to Reduce Platelet Hyperactivation.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm variant characterized by excessive production of platelets. Since the most common cause of mortality and morbidity in ET patients is thrombosis, the excessive production of platelets may cause thrombotic events. However, little is known about the function of platelets in ET. We report a female patient who presented as asymptomatic, without a remarkable medical history, and ET was diagnosed after an incidental finding of moderate thrombocytosis. Notably, together with thrombocytosis, an abnormal platelet phenotype was found for the presence of a massive, rapid and spontaneous formation of aggregates and platelet hypersensitivity to subthreshold concentrations of aggregating agonists. Bone marrow histopathological examination and genetic analysis with the JAK2 (V617F) gene mutation findings confirmed the initial suspicion of ET. Although the ET patient was placed on aspirin, the persistence of the platelet hyperactivation and hyperaggregability prompted a switch in antiplatelet medication from entero-coated (EC) to plain aspirin. As result, platelet hypersensitivity to agonists and spontaneous aggregation were no longer found. Collectively, our study demonstrates that platelet function analysis could be a reliable predictor of ET and that plain aspirin should be preferred over EC aspirin to attenuate platelet hyperreactivity.

Minimal residual disease by flow cytometry and allelic-specific oligonucleotide real-time quantitative polymerase chain reaction in patients with myeloma receiving lenalidomide maintenance: A pooled analysis.

BACKGROUND: Minimal residual disease (MRD) is one of the most relevant prognostic factors in patients with multiple myeloma (MM); however, the impact of maintenance therapy on MRD levels remains unclear. Among patients with newly diagnosed MM (NDMM) who received lenalidomide maintenance until they developed disease progression, the role of MRD status as a predictor of progression-free survival (PFS) was evaluated by multiparameter flow cytometry (MFC) and allelic-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) analysis. METHODS: Seventy-three patients with NDMM enrolled in the RV-MM-EMN-441 (clinical trials.gov identifier, NCT01091831) and RV-MM-COOP-0556 (clinicaltrials.gov identifier, NCT01208766; European Myeloma Network EMN02/HO95 MM Trial) phase 3 trials who achieved at least a very good partial response after intensification/consolidation were included. The median patient age was 57 years (interquartile range, 53-61 years), and all patients received lenalidomide maintenance until they developed progression. MRD was evaluated on bone marrow after intensification/consolidation, after 6 courses of maintenance, and every 6 months thereafter until clinical relapse using both ASO-RQ-PCR (sensitivity, 10-5 ) and MFC (sensitivity, from 10-4 to 10-5 ). RESULTS: After intensification/consolidation, 33 of 72 patients (46%) achieved a molecular complete response (m-CR), and 44 of 70 (63%) achieved a flow complete response (flow-CR). Almost 27% of patients who were MRD-positive after consolidation became MRD-negative during maintenance. After a median follow-up of 38 months, PFS was prolonged in patients who achieved negative MRD status during maintenance according to results from both ASO-RQ-PCR analysis (hazard ratio, 0.29; 95% confidence interval, 0.14-0.62; P = .0013) and MFC (hazard ratio, 0.19; 95% confidence interval, 0.09-0.41; P < .001). The impact of negative MRD status on PFS was similar in all subgroups (ASCT and no-ASCT; International Staging System stages I, II, and III; high-risk and standard-risk cytogenetics), and the two techniques were highly correlated. CONCLUSIONS: MRD status is a stronger predictor of PFS than standard risk factors, and lenalidomide maintenance further increases the rate of negative MRD results.

Highly sensitive MYD88L265P mutation detection by droplet digital polymerase chain reaction in Waldenström macroglobulinemia.

We here describe a novel method for MYD88L265P mutation detection and minimal residual disease monitoring in Waldenström macroglobulinemia, by droplet digital polymerase chain reaction, in bone marrow and peripheral blood cells, as well as in circulating cell-free DNA. Our method shows a sensitivity of 5.00×10-5, which is far superior to the widely used allele-specific polymerase chain reaction (1.00×10-3). Overall, 291 unsorted samples from 148 patients (133 with Waldenström macroglobulinemia, 11 with IgG lymphoplasmacytic lymphoma and 4 with IgM monoclonal gammopathy of undetermined significance) were analyzed: 194 were baseline samples and 97 were followup samples. One hundred and twenty-two of 128 (95.3%) bone marrow and 47/66 (71.2%) baseline peripheral blood samples scored positive for MYD88L265P To investigate whether MYD88L265P detection by droplet digital polymerase chain reaction could be used for minimal residual disease monitoring, mutation levels were compared with IGH-based minimal residual disease analysis in 10 patients, and was found to be as informative as the classical, standardized, but not yet validated in Waldenström macroglobulinemia, IGH-based minimal residual disease assay (r2=0.64). Finally, MYD88L265P detection by droplet digital polymerase chain reaction on plasma circulating tumor DNA from 60 patients showed a good correlation with bone marrow findings (bone marrow median mutational value 1.92×10-2, plasma circulating tumor DNA value: 1.4×10-2, peripheral blood value: 1.03×10-3). This study indicates that droplet digital polymerase chain reaction assay of MYD88L265P is a feasible and sensitive tool for mutation screening and minimal residual disease monitoring in Waldenström macroglobulinemia. Both unsorted bone marrow and peripheral blood samples can be reliably tested, as can circulating tumor DNA, which represents an attractive, less invasive alternative to bone marrow for MYD88L265P detection.

Minimal residual disease after transplantation or lenalidomide-based consolidation in myeloma patients: a prospective analysis.

We analyzed 50 patients who achieved at least a very good partial response in the RV-MM-EMN-441 study. Patients received consolidation with autologous stem-cell transplantation (ASCT) or cyclophosphamide-lenalidomide-dexamethasone (CRD), followed by Lenalidomide-based maintenance. We assessed minimal residual disease (MRD) by multi-parameter flow cytometry (MFC) and allelic-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) after consolidation, after 3 and 6 courses of maintenance, and thereafter every 6 months until progression. By MFC analysis, 19/50 patients achieved complete response (CR) after consolidation, and 7 additional patients during maintenance. A molecular marker was identified in 25/50 patients, 4/25 achieved molecular-CR after consolidation, and 3 additional patients during maintenance. A lower MRD value by MFC was found in ASCT patients compared with CRD patients (p=0.0134). Tumor burden reduction was different in patients with high-risk vs standard-risk cytogenetics (3.4 vs 5.2, ln-MFC; 3 vs 6 ln-PCR, respectively) and in patients who relapsed vs those who did not (4 vs 5, ln-MFC; 4.4 vs 7.8 ln-PCR). MRD progression anticipated clinical relapse by a median of 9 months while biochemical relapse by a median of 4 months. MRD allows the identification of a low-risk group, independently of response, and a better characterization of the activity of treatments.

Multiple myeloma: New surface antigens for the characterization of plasma cells in the era of novel agents.

BACKGROUND: Multiple Myeloma (MM) is a neoplastic disorder characterized by clonal proliferation of malignant plasma cells (PCs). Flow cytometry is an essential tool to confirm diagnosis and evaluate minimal residual disease (MRD). This study aims at identifying new surface PC markers suitable for targeted therapy in MM and able to improve MRD detection. METHODS: The expression of 82 molecules provided by the "Ninth International Workshop on Leukocyte Antigens" was analyzed by flow cytometry in 5 MM cell lines and in 20 newly diagnosed MM (NDMM) patients. Based on the antigens expression and monoclonal antibody availability, CD150, CD48, CD229, CD352, CD319, CD272, CD86, CD200 and CD184 were subsequently tested in 24 NDMM, 8 relapsed MM (RMM), 6 plasma cell leukemia (PCL) and 13 healthy subjects. RESULTS: CD352 was less frequently expressed on NDMM than on healthy PCs; CD200 was more frequently expressed on NDMM than on RMM and healthy PCs. CD150, CD319, CD229, CD352 Mean Fluorescence Intensity (MFI) was lower in pathological than in healthy samples. The proportion of CD150-positive samples was lower in NDMM and RMM than in healthy subjects; CD86+ samples were less frequent in NDMM than in healthy subjects; CD200+ samples were more frequent in NDMM than in RMM and healthy subjects. CONCLUSIONS: CD150, CD86 and CD200 can help to identify malignant PCs; CD272, CD319, CD229, CD48 are highly expressed on all PCs and could be considered for targeted therapy. All these antigens could be added to a routine panel for PCs identification and MRD evaluation.

The bone marrow of myeloma patients is steadily inhabited by a normal-sized pool of functional regulatory T cells irrespectiveof the disease status.

Conflicting data have been reported about the frequency and function of regulatory T cells in multiple myeloma. Most studies have investigated peripheral blood rather than bone marrow Tregs and side-by-side comparisons with bone marrow from healthy donors have still not been made. In this study, we show that regulatory T-cells total count, subset distribution, and expression of chemokine receptors are similar in the bone marrow of myeloma patients and healthy donors. Regulatory T cells are not recruited by myeloma cells in the bone marrow and their counts are unaffected by the tumor burden and the disease status. The diversity of T-cell receptor repertoire is highly preserved ensuring broad reactivity and effective suppressor function. Our results indicate that regulatory T cells may not be the main players of immunological tolerance to myeloma cells under base-line conditions, but their fully preserved immune competence may promote their inadvertent activation and blunt T-cell driven anti-myeloma immune interventions even after myeloma cells have successfully been cleared by chemotherapy.